shark_cloaca_17
Swabs were thawed at room temperature, removed from RNAlater solution, and each placed in a sterile 1.5 ml Eppendorf tube and air-dried for 10 min. DNA extraction was conducted using the Qiagen Blood & Tissue 96 kit according to manufacturers instructions (Qiagen, Germany). Briefly, 360 l of ATL buffer and 40 l of Proteinase K (at a final concentration of 20 mg/ml in a reaction volume of 400 l) were added per swab to the 1.5 ml tubes. The individual tubes containing swab samples in ATL buffer and Proteinase K were vortexed vigorously for 15 s and incubated at 56 C and 700 rpm for 90 min. Then, 1 l of RNase A was added 15 min before the end of the incubation. After the incubation, samples were vortexed again, the swabs removed, and lysates transferred to individual wells of a Qiagen Blood & Tissue 96-well plate. Volumes for buffer AL and for 70 % ethanol were adjusted to twice the volume (i.e., 400 l per reaction each). Further steps were followed as per the manufacturers instructions. All centrifugation steps were carried out at room temperature. Purified DNA bound to Qiagen spin column membranes was eluted once using 50 l of AE buffer. DNA was quantified using a Qubit 2-Fluorometer using the HS dsDNA kit (Invitrogen, USA). In addition to DNA extractions from samples, two types of mock DNA extractions were conducted as follows: 1) no template, using kit reagents only; 2) using a clean swab with kit reagents. For all samples, PCR amplifications were performed in triplicates using Qiagen Multiplex PCR Kit (Qiagen, Germany) with primers containing Illumina adapters (underlined below). For the 16S rRNA gene sequencing, we amplified the hypervariable regions V5 and V6 of the bacterial 16S rRNA gene. We used primers 16SMiSeqF-Andersson 5'TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGGATTAGATACCCTGGTA-3 and 16SMiSeqR-Andersson 5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCRRCACGAGCTGACGAC-3, which had previously been shown to amplify well with different marine templates (Bayer et al. 2013; Pogoreutz et al. 2018, 2019). Individual PCRs were run using 5 l Qiagen Mix, 0.3 l of each 10 M primer, 1 l of DNA template (1-2 ng of DNA per 1 l), and RNase-free water to adjust to a final reaction volume of 10 l. In addition to the samples, PCRs were run for templates from the mock DNA extractions, which were also subjected to sequencing. Thermal cycling conditions for 16S rRNA gene PCRs were: 95 C for 15 min, followed by 27 cycles of 95 C for 30 s, 55 C for 90 s, 72 C for 30 s, and a final extension cycle of 72 C at 10 min. Five l of each PCR product were run on an 1% agarose gel to visualize successful amplification. Sample triplicates were subsequently pooled and then purified with Illustra ExoProStar 1-Step (GE Healthcare Life Sciences, UK). Purified PCR products were subjected to an indexing PCR (8 cycles) to add Nextera XT indexing and sequencing adapters (Illumina, USA) according to the manufacturers protocol. Successful addition of indexes was confirmed by comparing the length of the initial PCR product to the corresponding indexed sample on a 1% agarose gel. Indexed PCR products were purified and normalized with the SequalPrep Normalization Plate Kit (Thermo Fisher Scientific, USA), followed by quantification on the BioAnalyzer (Agilent Technologies, USA) and QuBit (Quant-IT dsDNA High Sensitivity Assay Kit; Invitrogen, USA), and pooled in equimolar ratios. The library was sequenced at the Bioscience Core Lab (BCL) at King Abdullah University of Science and Technology (KAUST) at 15 pM with 10% phiX on an Illumina MiSeq, 2  300 bp end, Rapid run, 500 cycles, according to the manufacturers specifications. Overall, a total of 138 samples (15 seawater + 28 dorsal + 28 gill covering + 34 buccal + 33 cloacal samples) plus six blanks were prepared for sequencing.